The invention relates to Chlamydia pneumoniae antigenic polypeptides, fused proteins containing the polypeptides, DNAs coding therefore, recombinant vectors carrying the DNAS, transformants containing the recombinant vectors, a method for production of antibody, a method and reagents for detection and/or measurement of antibody, a method and agents for diagnosis of Chlamydia pneumoniae infections, probes and primers for detection and/or measurement of Chlamydia pneumoniae gene, and a method and reagents for detection and/or measurement of Chlamydia pneumoniae gene. The invention can be effectively used in the pharmaceutical industry, particularly in the preparation of agents for diagnosis of Chlamydia pneumoniae infections.
Several kinds of species are known in Chlamydia, that is, Chlamydia trachomatis, Chlamydia psittaci, Chlamydia pecorum, Chlamydia pneumoniae and the like. Chlamydia trachomatis causes trachoma, venereal lymphogranuloma, urogenital infections, inclusion conjunctivitis, neonatal pneumonia and the like. Chlamydia psittaci causes psittocosis and the like. Chlamydia pneumoniae causes respiratory infections, atypical pneumonia and the like.
Since the symptoms of infections in the respiratory apparatus which are caused by Chlamydia pneumoniae are similar to those of infections caused by Mycoplasma pneumoniae or Influenza virus, physicians often make a wrong diagnosis. Hence, there is a need for the development of a simple method for diagnosing the infections caused by Chlamydia pneumoniae. 
In general, an infection can reliably be diagnosed by detecting the causative bacterium in the infected site or by detecting an antibody against the causative bacterium in body fluids such as a sera and the like. The former method is called an antigen test and the latter is called an antibody test. Both of them are clinically important. As for Chlamydia pneumoniae, there is known an antibody test which is carried out by a method in which an antibody is detected by using an elementary body of Chlamydia pneumoniae. 
However, this method has the disadvantage that the elementary body of Chlamydia pneumoniae reacts not only with an antibody against Chlamydia pneumoniae but also with antibodies against other species of Chlamydia, thus being fairly unspecific. This is because the elementary body of Chlamydia pneumoniae contains an antigen which is also present in other species of geneus Chlamydia than Chlamydia pneumoniae, that is, Chlamydia trachomatis and Chlamydia psittaci. 
As a plasmid which can be used for the expression of a large amount of a protein in E. coli, pBBK10MM is known (Japanese Unexamined Patent Publication No. Hei 4-117284). This plasmid can be used for the expression of a fused protein of an anti-allergic peptide with DHFR. The expressed fused protein also maintains the enzymatic activity of DHFR and can therefore be purified easily by utilizing the characteristic properties and activities of DHFR.
Genetic screening has been carried out to diagnose infections. In this screening, the presence of the gene of a microorganism to be detected in a sample is examined using nucleic acid probes and the like.
As for Chlamydia pneumoniae, there is known a genetic screening method which is carried out as disclosed in Japanese Unexamined Patent Publication No. Sho 64-500083, U.S. Pat. No. 5,281,518 and WO94/04549.
However, Japanese Unexamined Patent Publication No. Sho 64-500083 and U.S. Pat. No. 5,281,518 only disclose that a chromosomal DNA of Chlamydia pneumoniae or a DNA fragment which is obtained by cleaving the chromosomal DNA with a restriction enzyme or the like is used as a probe. The base sequences of these DNA molecules are not determined and the specificity of these probes are therefore unclear. In addition, it is difficult to determine the reaction conditions.
Although WO94/04549 discloses a method using a probe which is hybridized to ribosome RNA or DNA corresponding thereto, the specificity of these probes is not reliable because the homology of ribosomal RNA is relatively high in all organisms.
It is an object of the invention to provide antigenic polypeptides that do not react with antibodies against species of geneus Chlamydia other than Chlamydia pneumoniae, such as Chlamydia trachomatis, Chlamydia psittaci and the like and which react only with a Chlamydia pneumoniae-specific antibody and can thereby detect the Chlamydia pneumoniae-specific antibody.
Another object of the invention is to provide a method for synthesizing large amounts of the antigenic polypeptides by using gene recombination techniques.
A further object of the invention is to provide a method for production of an anti-Chlamydia pneumoniae-specific antibody, a method and reagents for detection and/or measurement of the anti-Chlamydia pneumoniae-specific antibody, and agents for diagnosis of Chlamydia pneumoniae infections, all by using said antigenic polypeptides.
A still further object of the invention is to provide probes and primers for detecting and/or measuring specifically Chlamydia pneumoniae gene, a method and reagents for detection and/or measurement of Chlamydia pneumoniae gene and agents for diagnosis of Chlamydia pneumoniae infections, all by using the probes or primers.
An even further object of the invention is to provide antigenic polypeptides for detection of an antibody which reacts with geneus Chlamydia including Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydia psittaci and the like.
The subject matters of the invention are as follows:
(1) A Chlamydia pneumoniae antigenic polypeptide, which comprises polypeptide containing a sequence of at least 5 consecutive amino acids in the polypeptide of SEQ ID NO: 1 (hereinafter referred to as xe2x80x9cpolypeptide Axe2x80x9d).
(2) The antigenic polypeptide of (1), wherein said polypeptide A is a polypeptide in which at least one amino acid is deleted from the polypeptide of SEQ ID NO: 1.
(3) The antigenic polypeptide of (1), wherein said polypeptide A is a polypeptide in which at least one amino acid in the polypeptide of SEQ ID NO: 1 is replaced with other amino acid or a polypeptide in which at least one amino acid is added in the polypeptide of SEQ ID NO: 1.
(4) The antigenic polypeptide of (1), wherein said polypeptide A is a polypeptide in which an amino acid or a peptide sequence is bound to a sequence of at least 5 consecutive amino acids in the polypeptide of SEQ ID NO: 1.
(5) The antigenic polypeptide of (1), wherein said polypeptide A is a polypeptide containing the amino acid sequence of SEQ ID NO: 1.
(6) The antigenic polypeptide of (1), wherein said polypeptide A is a polypeptide containing the amino acid sequence of SEQ ID NO: 2.
(7) The antigenic polypeptide of (1), wherein said polypeptide A is a polypeptide containing the amino acid sequence of SEQ ID NO: 5.
(8) A DNA encoding the antigenic polypeptide of any one of (1)-(7), or a DNA complementary thereto.
(9) The DNA of (8), which contains the base sequence of SEQ ID NO: 3.
(10) The DNA of (8), which contains the base sequence of SEQ ID NO: 4.
(11) The DNA of (8), which contains the base sequence of SEQ ID NO: 7.
(12) A recombinant vector carrying the DNA of any one of (8)-(11).
(13) The recombinant vector of (12), which is plasmid pCPN533 xcex1 containing the base sequence of SEQ ID NO: 10.
(14) A transformant containing the recombinant vector of (12) or (13).
(15) A method for production of an anti-Chlamydia pneumoniae antibody,
wherein the antigenic polypeptide of any one of (1)-(7) is used as an antigen.
(16) A method for detection and/or measurement of an anti-Chlamydia pneumoniae antibody, wherein the antigenic polypeptide of any one of (1)-(7) is used as an antigen.
(17) A reagent for detection and/or measurement of an anti-Chlamydia pneumoniae antibody, which comprises the antigenic polypeptide of any one of (1)-(7) as an antigen.
(18) A reagent for diagnosis of a Chlamydia pneumoniae infection, which comprises the antigenic polypeptide of any one of (1)-(7) as an active ingredient.
(19) A fused protein of a Chlamydia pneumoniae antigenic polypeptide with dihydrofolate reductase, in which polypeptide containing a sequence of at least 5 consecutive amino acids in the polypeptide of SEQ ID NO: 1 is bound to the polypeptide of SEQ ID NO: 14 (hereinafter referred to as xe2x80x9cpolypeptide Bxe2x80x9d) either directly or via an intervening amino acid or amino acid sequence.
(20) The fused protein of (19), wherein said polypeptide B is a polypeptide in which at least one amino acid is deleted from the polypeptide of SEQ ID NO: 1.
(21) The fused protein of (19), wherein said polypeptide B is a polypeptide in which at least one amino acid in the polypeptide of SEQ ID NO: 1 is replaced with other amino acids or a polypeptide in which at least one amino acid is added in the polypeptide of SEQ ID NO: 1.
(22) The fused protein of (19), which is a polypeptide containing the amino acid sequence of SEQ ID NO: 15.
(23) The fused protein of (19), which is a polypeptide containing the amino acid sequence of SEQ ID NO: 16.
(24) A DNA encoding the fused protein of any one of (19)-(23), or a DNA complementary thereto.
(25) The DNA of (24), which contains the base sequence of SEQ ID NO: 17.
(26) The DNA of (24), which contains the base sequence of SEQ ID NO: 18.
(27) A recombinant vector carrying the DNA of any one of (24)-(26).
(28) The recombinant vector of (27), which is plasmid pCPN533T.
(29) A transformant containing the recombinant vector of (27) or (28).
(30) A method for production of an anti-Chlamydia pneumoniae antibody, wherein the fused protein of any one of (19)-(23) is used as an antigen.
(31) A method for detection and/or measurement of an anti-Chlamydia pneumoniae antibody, wherein the fused protein of any one of (19)-(23) is used as an antigen.
(32) A reagent for detection and/or measurement of an anti-Chlamydia pneumoniae antibody, which comprises the fused protein of any one of (19)-(23) as an antigen.
(33) A reagent for diagnosis of a Chlamydia pneumoniae infection, which comprises the fused protein of any one of (19)-(23) as an active ingredient.
(34) A probe for detection and/or measurement of Chlamydia pneumoniae gene, which comprises any one of
(a) a DNA containing a sequence of at least 10 consecutive bases in the DNA of SEQ ID NO: 3,
(b) a DNA complementary to DNA (a), or
(c) a DNA having at least 90% homology to DNA (a) or (b).
(35) The probe of (34), which contains the base sequence of SEQ ID NO: 19.
(36) The probe of (34), which contains the base sequence of SEQ ID NO: 20.
(37) A method for detection and/or measurement of Chlamydia pneumoniae gene, characterized in that the probe of any one of (34)-(36) is used.
(38) A reagent for detection and/or measurement of Chlamydia pneumoniae gene, which comprises the probe of any one of (34)-(36).
(39) An agent for diagnosis of a Chlamydia pneumoniae infection, which comprises the probe of any one of (34)-(36) as an active ingredient.
(40) A primer for detection and/or measurement of Chlamydia pneumoniae gene, which comprises any one of
(a) a DNA containing a sequence of at least 10 consecutive bases in the DNA of SEQ ID NO: 3,
(b) a DNA complementary to DNA (a), or
(c) a DNA having at least 90% homology to DNA (a) or (b).
(41) The primer of (40), which contains the base sequence of SEQ ID NO: 19.
(42) The primer of (40), which contains the base sequence of SEQ ID NO: 20.
(43) A method for detection and/or measurement of Chlamydia pneumoniae gene, wherein the primer of any one of (40)-(42) is used.
(44) A reagent for detection and/or measurement of Chlamydia pneumoniae gene, which comprises the primer of any one of (40)-(42).
(45) A reagent for diagnosis of a Chlamydia pneumoniae infection, which comprises the primer of any one of (40)-(42) as an active ingredient.
(46) A Chlamydia pneumoniae antigenic polypeptide, which is selected from the group consisting of
(a) the polypeptide of SEQ ID NO: 5,
(b) a polypeptide in which at least one amino acid is deleted from the polypeptide of SEQ ID NO: 5,
(c) a polypeptide in which at least one amino acid in the polypeptide of SEQ ID NO: 5 is replaced with another amino acid, and
(d) a fused polypeptide of any one of (a)-(c) with another amino acid or peptide.
(47) A Chlamydia pneumoniae antigenic polypeptide, which is selected from the group consisting of
(a) the polypeptide of SEQ ID NO: 6,
(b) a polypeptide in which at least one amino acid is deleted from the polypeptide of SEQ ID NO: 6,
(c) a polypeptide in which at least one amino acid in the polypeptide of SEQ ID NO: 6 is replaced with another amino acid, and
(d) a fused polypeptide of any one of (a)-(c) with another amino acid or peptide.
(48) A DNA encoding the polypeptide of (46), or a DNA complementary thereto.
(49) A DNA encoding the polypeptide of (47), or a DNA complementary thereto.
(50) The DNA of (48), wherein said DNA encoding the polypeptide of (46) is the DNA of SEQ ID NO: 7.
(51) The DNA of (49), wherein said DNA encoding the polypeptide of (47) is the DNA of SEQ ID NO: 8.
(52) A recombinant vector carrying the DNA of any one of (48)-(51).
In the specification, deoxynucleotides having only one base are referred to as xe2x80x9cmonodeoxynucleotidesxe2x80x9d and deoxynucleotides having at least two bases are referred to as xe2x80x9cDNAsxe2x80x9d, unless otherwise indicated.
The invention will now be explained in detail.
The antigen polypeptide of the present invention is formed of polypeptides containing at least five continued amino acid sequences in a polypeptide of SEQ ID No. 1 (hereinafter referred to as xe2x80x9cPolypeptide Axe2x80x9d) from the viewpoint of the minimum size in which a peptide is allowed to possess antigenicity.
Since the antigen-antibody reaction can be expected to gain in sensitivity in proportion as the length of amino acid sequence. increases, the polypeptide A is appropriately formed of not less than 20, preferably not less than 100, and more preferably not less than 250 amino acids.
So long as the polypeptide A possesses the antigenicity inherent in Chlamydia pneumoniae, it tolerates the loss of amino acids (1-250 amino acids, for example) from the polypeptide of SEQ ID No. 1. If the number of missing amino acids is unduly large, the polypeptide A will tend to suffer the antigenicity inherent in Chlamydia pnuemoniae to be impaired.
When the number of missing amino acids is large (five or more, for example), the polypeptide A prefers such missing amino acids (five or more, for example) to occur in a continued series for the sake of retaining the antigenicity of Chlamydia pneumoniae. 
So long as the polypeptide A possesses the antigenicity inherent in Chlamydia pneumoniae, it tolerates the substitution of part of the amino acids (1-100 amino acids, for example) by other amino acids or the insertion of amino acids (1-100 amino acids, for example) in the polypeptide of SEQ ID No. 1. If the number of amino acids involved in the substitution or insertion is unduly large, the polypeptide A will tend to suffer the antigenicity inherent in Chlamydia pnuemoniae to be impaired. When the number of amino acids involved in the substitution or insertion is large (five or more, for example), the polypeptide A prefers the amino acids (five or more, for example) to occur in a continued series for the sake of retaining the antigenicity of Chlamydia pneumoniae. The amino acids to be involved in the substitution are preferred to possess such similar qualities as are observed in the substitution between glycine and alanine, for example.
So long as the polypeptide A possesses the antigenicity inherent in Chlamydia pneumoniae, it may be a polypeptide having amino acids or peptides ligated directly or through the medium of an intervening amino acid sequence to at least five continued amino acid sequences in the polypeptide of SEQ ID No. 1.
The peptides for the ligation are appropriately formed of not more than 1000 amino acid sequences, preferably not more than 500 amino acid sequences, and more preferably not more than 200 amino acid sequences for the sake of retaining the antigenicity inherent in Chlamydia pneumoniae. 
As concrete examples of such amino acids or peptides, leucine, leucine-methionine, dihydrofolic acid reductase (DHFR), and xcex2-galactosidase may be cited.
As concrete examples of the polypeptide A using DHFR or xcex2-galactosidase as a peptide, DHFR-Chlamydia pneumoniae antigen polypeptide-fused protein and xcex2-galactosidase-Chlamydia pneumoniae antigen polypeptide-fused protein may be cited. DHFR or xcex2-galactosidase may be ligated either directly or through the medium of an intervening amino acid sequence with Chlamydia pneumoniae antigen polypeptide.
As concrete examples of the polypeptide A, the polypeptides of SEQ ID No. 1, SEQ ID No. 2, and Sequence No. 5 may be cited.
Though the intervening amino acid sequence is not defined particularly, the amino acid sequences of leucine and leucine-methionine are examples.
As concrete examples of the fused protein of the present invention, the polypeptide formed of amino acid sequences of SEQ ID No. 15 and the polypeptide formed of amino acid sequences of SEQ ID No. 16 may be cited.
Among the fused proteins cited above, the polypeptide formed of the amino acid sequences of SEQ ID No. 15 including the whole antigen polypeptide of 53 kDa of Chlamydia pneumoniae proves particularly advantageous.
The method of chemical synthesis and the method of gene recombination are available for the production of the antigen polypeptide of this invention.
The polypeptide of SEQ ID No. 1 of this invention is an antigen polypeptide formed of 488 amino acid residues as shown in the table of sequences.
The polypeptide of SEQ ID No. 2 of this invention is an At antigen polypeptide formed of 271 amino acid residues as shown in the table of sequences.
The polypeptide of SEQ ID No. 5 of this invention is an antigen polypeptide formed of 259 amino acid residues as shown in the table of sequences.
Among other antigen polypeptides mentioned above, the polypeptide of SEQ ID No. 1 containing the whole antigen polypeptide of 53 kDa of Chlamydia pnuemoniae proves particularly advantageous.
The method of chemical synthesis and the method of gene recombination are available for the production of the antigen polypeptide of this invention.
Among the methods of chemical synthesis is counted the MAP (multiple antigen peptide) method, for example. The MAP method befits the synthesis of a peptide formed of not more than 30 amino acid sequences. This synthesis can be implemented by the use of a commercially available peptide synthesizing device.
Among the methods of gene recombination is counted a method which comprises inserting a DNA coding for the antigen polypeptide of this invention in a vector thereby constructing a recombinant vector, inserting the recombinant vector in a host thereby producing a transformant, and isolating the peptide aimed at from the transformant.
The DNA coding for the antigen polypeptide of this invention will be described afterward.
The vector may be plasmid, phage, etc.
As concrete examples of the host, Escherichia coli, Bacillus subtilis, yeast, etc. may be cited.
Now, the method for forming the transformant and the method for refining the peptide aimed at by the use of the transformant will be described in detail below.
The xcex phage obtained by screening (see infra) is already a kind of recombinant vector carrying the DNA of the invention. Additional recombinant vectors can be prepared by inserting in a known plasmid vector or phage vector the DNA encoding the Chlamydia pneumoniae antigenic polypeptide (see infra) in a conventional procedure. In this case, a linker may be used if necessary. As the known plasmid vector, pBR322, pUC18, pUC19, pBBK10MM or the like can be used. Plasmids pBR322, pUC18 and pUC19 are commercially available and pBBK10MM is described in detail in Japanse Unexamined Patent Publication No. Hei 4-117284. As the phage vector, xcex gt11 phage, xcex gt10 phage or the like can be used. In any case, recombinant vectors corresponding to the parent vectors used can be obtained.
The recombinant vectors carrying the DNA of the invention include plasmid pCPN533 xcex1, 53-3S xcex phage and the like (see infra).
The obtained recombinant vector is introduced into a host to prepare a transformant. If an E. coli-derived plasmid or xcex phage is used, an E. coli strain such as HB 101 can be used as a host. The host is treated to become a competent cell. A competent cell obtained by treating E. coli strain HB101 is commercially available from Takara Shuzo Co., Ltd. A method of introducing the recombinant vector into a host to prepare a transformant is described in xe2x80x9cMolecular Cloningxe2x80x9d.
The obtained transformant is cultured to form colonies. Plasmid DNAs are obtained from each of the colonies and cleaved with an appropriate restriction enzyme. A transformant having a desired recombinant plasmid is selected according to the results of agarose gel electrophoretic analysis of the cleaved plasmid DNA. The plasmid vectors thus prepared include plasmid pCPN533 xcex1.
Examples of the transformant thus prepared include E. coli strain HB101 containing the recombinant vector pCPN533 xcex1.
The DNA molecule encoding the Chlamydia pneumoniae antigenic polypeptide (see infra) is ligated to the DNA molecule encoding DHFR (see infra) by means of a commercially available kit. In the ligation, a linker may be used if necessary. A DNA ligation kit (Takara Shuzo Co., Ltd) can be used as a commercially available kit. If the DNA obtained by the ligation does not have a replication origin and does not therefore function as a plasmid, the DNA is inserted in a separate plasmid vector, which may be pBR322, pUC18 or the like.
The ligated DNA is introduced into a host to prepare a transformant. If an E. coli-derived plasmid is used, an E. coli strain such as HB 101 can be used as a host. The host is treated to become a competent cell. A competent cell obtained by treating E. coli strain HB101 is commercially available from Takara Shuzo Co., Ltd. The method of introducing the ligated DNA into a host to prepare a transformant is described in xe2x80x9cMolecular Cloningxe2x80x9d.
The obtained transformant is cultured to form colonies. Plasmid DNAs are obtained from each of the colonies and cleaved with an appropriate restriction enzyme. A transformant having a desired recombinant plasmid is selected according to the results of agarose gel electrophoretic analysis. An example of the plasmid vector thus prepared is plasmid pCPN533T.
An example of the transformant thus prepared is E. coli strain HB101 containing the recombinant vector pCPN533T.
The transformant is cultured by shaking an incubator containing the transfomant at an appropriate temperature in a medium that allows the transformant to grow until a sufficient amount of the desired antigenic polypeptide is accumulated in the transformant. If E. coli strain HB101 containing the recombinant vectors pCPN533 xcex1 or pCPN533T are used as a transformant, the cell is cultured while shaking in ampicillin-containing LB medium at 37xc2x0 C. overnight. Subsequently, the culture is inoculated in ampicillin-containing TB medium and further cultured while shaking at 37xc2x0 C. overnight. A method for preparing the TB medium is described in xe2x80x9cMolecular Cloningxe2x80x9d.
The cultured transformant is harvested by centrifugation and suspended in a buffer. The transformant is disrupted by sonication of the suspension. If the transformant is E. coli, the cell may be lysed by successively adding lysozyme and an SDS-containing buffer to the suspension.
When the polypeptide aimed at is secretory in quality, the culture broth is centrifuged to obtain the supernatant.
After the disruption of the transformant, the cell residue is removed by centrifugation, thereby obtaining the supernatant. Streptomycin sulfate is added to the supernatant. The mixture is stirred for a certain period of time and centrifuged to precipitate nucleic acids, thereby obtaining the supernatant.
This supernatant is precipitated with ammonium sulfate and centrifuged. Generally, the precipitate is recovered as the product. Since the supernatant possibly contains the peptide aimed at, the practice of sampling and analyzing the supernatant thereby confirming the presence or absence of the peptide proves advantageous.
Either the solution of the precipitate in a small amount of buffer solution or the supernatant is fractionated by liquid chromatography. The proteins contained in the fractions are blotted by the Western blotting method using a Chlamydia pneumoniae-specific monoclonal antibody to obtain the fractions containing antigen polypeptide. When the polypeptide A is a protein fused with DHFR, a Methotrexate column can be used as the column for the liquid chromatography. Specific procedures of the removal of residues such as a cell membrane and the like, the removal of DNA by addition of streptomycin sulfate, the recovery of proteins by addition of ammonium sulfate and a Western blotting method are described in xe2x80x9cMolecular Cloningxe2x80x9d.
In the invention, the DNA encoding the polypeptide of SEQ ID NO: 1 means DNAS selected from the group of DNAs which are obtained by translating the amino acids of the polypeptide of SEQ ID NO: 1 to triplets in accordance with the genetic code (each amino acid is assigned 1-6 sets of nucleotide sequences). This group of DNAs includes the DNA of SEQ ID NO: 3.
The DNA encoding the antigenic polypeptide A means DNAs encoding the polypeptide A. These DNAs are selected from the group of DNAs which are obtained by translating the amino acid sequence for the polypeptide A to triplets in accordance with the genetic code.
As the polypeptide A, those polypeptides which have been described under the item xe2x80x9cAntigenic Polypeptidesxe2x80x9d above may be given. As the DNA encoding the polypeptide A, nucleotides sequences which correspond to the amino acid sequences for those polypeptides may be given.
Similarly, the DNA encoding the polypeptide of SEQ ID NO: 2 means DNAs selected from the group of DNAs which are obtained by translating the amino acids of the polypeptide of SEQ ID NO: 2 to triplets in accordance with the genetic code. This group of DNAS includes the DNA of SEQ ID NO: 4.
Additionally, the DNA encoding the polypeptide of SEQ ID NO: 5 means DNAs selected from the group of DNAS which are obtained by translating the amino acids of the polypeptide of SEQ ID NO: 5 to triplets in accordance with the genetic code. This group of DNAs includes the DNA of SEQ ID NO: 7.
Moreover, the DNA encoding the polypeptide of SEQ ID NO: 6 means DNAs selected from the group of DNAs which are obtained by translating the amino acids of the polypeptide of SEQ ID NO: 6 to triplets in accordance with the genetic code. This group of DNAs includes the DNA of SEQ ID NO: 8.
DNAs encoding the fused proteins comprise codons corresponding to the amino acid sequence of the fused protein. The DNAs include but are not limited to the DNAs of SEQ ID NOs: 17 and 18.
The base sequence of SEQ ID No. 17 is the base sequence of the DNA coding for the fused protein of DHFR and the whole antigen polypeptide of 53 kDa of Chlamydia pneumoniae and the base sequence of SEQ ID No. 18 is the base sequence of the DNA coding for the fused protein of DHFR and (part of) the antigen polypeptide of 53 kDa of Chlamydia pneumoniae. 
These DNA""s can be manufactured by the method of chemical synthesis or the method of gene recombination.
Among the methods of chemical synthesis is counted the phosphoamidite method which fits the synthesis of a DNA formed in a length of not more than 100 base sequences. This chemical synthesis can be attained by a commercially available DNA synthesizing device.
Among the methods of gene recombination are counted a method for cloning the DNA from the elementary body of Chlamydia pneumoniae in the manner already described and the PCR method utilizing the already acquired DNA as a template and using a primer manufactured by adopting the base sequence at a position arbitrarily selected in that DNA. The method of gene recombination is capable of manufacturing a long DNA of more than 100 bases.
Now, the method for cloning the DNA coding for the antigen polypeptide from the elementary body of Chlamydia pneumoniae will be described in detail below.
A suspension of cells is prepared from cultured HL cells. The supernatant of the culture is removed and the suspension of Chlamydia pneumoniae is then added to the resulting cell sheet. After incubation, Chlamydia pneuminiae-infected HL cells are obtained by centrifugation. As Chlamydia pneumoniae , strain YK41 (Y. Kanamoto et al., Micro biol. Immunol., Vol. 37, p.495-498, 1993) can be used.
The Chlamydia pneuminiae-infected HL cells are disrupted and centrifuged, thereby recovering the supernatant. The obtained supernatant is layered onto a continuous density gradient solution containing Urografin (Schering) is centrifuged.
The yellowish white band was recovered because in the preliminary experiment, it was confirmed to contain the elementary body of Chlamydia pneumoniae with the aid of an electron microscope.
The elementary body of Chlamydia pneumoniae is suspended in 10 mM Tris-HCl buffer (pH 8.0) containing 1 mM ethylene diaminetetra acetate (EDTA) (hereinafter referred to as xe2x80x9cTE bufferxe2x80x9d). To the resulting suspension are added a 1% aqueous solution of sodium dodecyl sulfate (SDS) and an aqueous solution of Proteinase K (1 mg/ml) and the elementary body is lysed while incubating. To the resulting solution is added phenol saturated with 0.1 M Tris-HCl buffer (pH 8.0). The mixture is stirred and centrifuged to recover an aqueous layer. The obtained aqueous layer is treated successively with RNase and phenol/chloroform/isoamyl alcohol, followed by ethanol precipitation. As a result, genomic DNA of Chlamydia pneunomiae is obtained.
The genomic DNA is digested with restriction enzymes AccI, HaeIII and AluI. The digest is treated with phenol/chloroform/isoamyl alcohol and subjected to ethanol precipitation to yield partially digested DNAs. To the partially digested DNAs are added a linker, adenosine 5xe2x80x2-triphosphate (hereinafter abbreviated to xe2x80x9cATPxe2x80x9d) and T4 ligase, thereby ligating the linker to the partially digested DNAs.
The linker-ligated partially digested DNAs are applied to a Chroma spin 6000 column in which the mobile phase is 10 mM Tris-HCl buffer containing 0.1 M NaCl and 1 mM EDTA. The eluate is collected and fractions containing 1-7 kbp DNA fragments are recovered. To the resulting fractions are added ATP and T4 polynucleotide kinase and a reaction is conducted to phosphorylate the 5xe2x80x2 end of the DNA fragments. The reaction solution is treated with phenol/chloroform/isoamyl alcohol and subjected to ethanol precipitation to yield 5xe2x80x2-end-phosphorylated DNA fragments.
To the resulting DNA fragments are added xcex gt11 DNA preliminarily digested with restriction enzyme EcoRI, ATP and T4 ligase and a reaction is conducted. The resulting recombinant xcex gt11 DNA is packaged with a commercially available packaging kit to prepare a gemonic DNA expression library.
Cultured cells of E. coli strain Y1090r- are infected with the gemonic DNA expression library and incubated in an agar medium. A protein produced in the cells by the expression of the inserted DNA is transferred to a nitrocellulose filter immersed in an aqueous solution of isopropylthio-xcex2-D-galactoside (IPTG). The filter is blocked with a bovine serum albumin and washed. The filter is then reacted with a Chlamydia pneumoniae-specific monoclonal antibody. As the Chlamydia pneumoniae-specific monoclonal antibody, AY6E2E8 and SCP53 can be used. A hybridoma cell line forming AY6E2E8 has been deposited with the National Institute of Bioscience and Human-Technology, the Agency of Industrial Science and Technology (1-3, Higashi 1 chome Tsukuba-shi Ibaraki-ken 305, Japan) as FERM BP-5154 under the terms of the Budapest Treaty. A hybridoma cell line forming SCP53 is disclosed in J. Clin. Microbil., Vol.132, p.583-588, 1994. After the reaction, the filter is washed and reacted with an anti-mouse IgG antibody labeled with an enzyme such as peroxidase or the like. After the reaction, the filter is washed and reacted with a color-developing substrate solution. As the color-developing substrate solution, a mixture of an aqueous solution of hydrogen peroxide and a solution of 4-chloro-1-naphthol in methanol can be used. After the reaction, the filter is washed and dried in air.
Plaques corresponding to the color-developing spots on the filter are identified and xcex phage contained in the plaques is obtained. The above procedure is repeated until all the plaques react with the aforementioned monoclonal antibody. As a result, the DNA encoding an antigenic polypeptide is cloned and xcex phage expressing the Chlamydia pneumoniae-specific antigenic polypeptide having reactivity with the Chlamydia pneumoniae-specific monochonal antibody is obtained.
E. coli strain Y109Or- is infected with the obtained xcex phage and cultured to yield a large amount of xcex phage. DNA molecules are obtained and purified from the xcex phage using a commercially available kit. To the obtained DNA molecules are added a primer, Taq polymerase and deoxynucleotides. The steps of heating, cooling and incubating are repeated, thereby amplifying the DNA molecule inserted in xcex gt11. xcex gt11 forward primer and xcex gt11 reverse primer (Takara Shuzo Co. Ltd.) can be used as primers and AmpliTaq DNA polymerase can be used as a Taq polymerase. A general procedure of DNA amplification is known as the PCR method, which is described in detail in J. Sambrook et al., Molecular Cloning, 2nd ed., Cold Spring Harbor Laboratory Press (1989) (hereinafter referred to as xe2x80x9cMolecular Cloningxe2x80x9d).
The amplified DNA is obtained and its base sequence is determined and analyzed. The amplified DNA can be obtained with a commercially available kit such as Wizard PCR Prep kit (Promega). The base sequence can be determined by fluorescence-labeled terminator cycle sequencing using Taq polymerase. This sequencing can be performed with a kit commercially available from Perkin-Elmer Japan. For analysis of the base sequence, a commercially available apparatus such as Model 373A DNA Sequencer (Applied Biosystelus) can be used.
Following the determination of the base sequence, the base sequence of the DNA is analyzed using a DNA sequencing software package such as DNASIS (Hitachi Software Engineering) to estimate an editing, junctional and amino acid-translational regions.
If it is found that a full-length gene has not been obtained, DNA molecules upstream and downstream of the available DNA are obtained by genome walking. The genome walking can be performed with a kit commercially available from Takara Shuzo Co., Ltd.
DNA encoding DHFR is obtained by digesting the DNA with a restriction enzyme from a plasmid vector containing the DNA or by amplifying the DNA by PCR using a template plasmid DNA or genomic DNA containing the DNA with an appropriate primer.
In the former method, plasmid vector pBBK10MM and recombinant vector pCPN533T of the invention can be used as the plasmid vector containing DNA encoding DHFR. E. coli containing pCPN533T and E. coli containing pBBK10MM have been deposited with the National Institute of Bioscience and Human-Technology, the Agency of Industrial Science and Technology as FERM BP-5222 and FERM BP-2374, respectively. Plasmid pCPN533T can be obtained from the deposited E. coli by a conventional method for obtaining plasmid DNA, which is described in xe2x80x9cMolecular Cloningxe2x80x9d. When plasmid pBBK10MM is used, a DNA fragment having a length of about 4.8 kbp may be excised with restriction enzymes BamHI and XhoI.
In the latter method, pBBK10MM and pCPN533T (see supra) can, be used as a plasmid DNA and genomic DNA of Bacillus subtilis can be used as a genomic DNA. Genomic DNA can be obtained by a conventional method for obtaining gemonic DNA, which is described in xe2x80x9cMolecular Cloningxe2x80x9d.
The primer to be used in the latter method can be designed and synthesized in consideration of base sequences at the 5xe2x80x2 and 3xe2x80x2 ends of DNA encoding DHFR. For example, an oligonucleotide having the 1-20 sequence in the base sequence of SEQ ID NO: 17 and one having a sequence complementary to the 461-480 sequence in the base sequence of SEQ ID NO: 5 can be used. These oligonucleotides can be synthesized chemically with a commercially available DNA synthesizer.
In the antigen polypeptides mentioned above, the polypeptide of SEQ ID NO. 1 containing the whole antigen polypeptide of 53 kDa of Chlamydia pneumoniae is particularly preferred.
An anti-Chlamydia pneumoniae antibody can be produced by immunizing a mouse with the antigenic polypeptide of the invention as an antigen, separating a spleen cell from the immunized mouse, fusing the spleen cell with a myeloma cell line to produce hybridomas, selecting a hybridoma recognizing the Chlamydia pneumoniae 53 kDa antigenic polypeptide from the produced hybridomas and culturing the selected hydridoma.
Exemplary myeloma cell lines include P3X63Ag8.653 (ATCC CRL-1580) and P3/NSI/1-Ag4-1 (ATCC TIB-18).
The anti-Chlamydia pneumoniae antibody is produced by a known general procedure for obtaining antibodies by immunization of mouse, except that the antigenic polypeptide of the invention is used as an antigen. Method and Reagents for Detection and/or Measurement of Anti-Chlamydia pneumoniae Antibody Using the Antigenic Polypeptide as Antigen, and Agents for Diagnosis of Chlamydia pneumoniae Infections Comprising the Antigenic Polypeptide as Active Ingredient
A method for detection and/or measurement of an anti-Chlamydia pneumoniae antibody comprises, for example, the steps of immobilizing the antigenic polypeptide on a support, applying a sample, washing, adding a labeled secondary antibody, washing and detecting and/or measuring the label either directly or indirectly.
Examples of the support include latex particles, cellulose threads, plastic assay plates and particles and the like.
The antigenic polypeptide may be immobilized on the support through covalent bonding or physical adsorption.
Examples of the sample include human sera and the like. It is preferred to block the surface of the support with bovine serum albumin or the like before the addition of a sample so as to insure that other antibodies in the sample will not bind to the support unspecifically.
The support is washed with a surfactant-containing phosphate buffer or the like.
An example of the labeled secondary antibody is a labeled anti-human monoclonal antibody. Useful labels include various kinds of enzymes such as alkaline phosphatase, luciferase, peroxidase, xcex2-galactosidase and the like, various fluorescent compounds such as fluorescein and the like. A chemical compound such as biotin, avidin, streptoavidin, digoxigenin or the like may be inserted between the antibody and the label.
When the label is an enzyme, it may be detected and/or measured by adding a substrate and detecting and/or measuring the light emission or color development which occurs due to the catalytic action of the enzyme or by measuring the change in light absorbance. When the label is a fulorescent compound, it may be detected and/or measured by irradiating the reaction system with UV light and detecting and/or measuring the emitted fluorescence. A sensitizer may be used if necessary.
Reagents for detection and/or measurement of the anti-Chlamydia pneumoniae antibody using the antigenic polypeptide of interest as an antigen include the antigenic polypeptides which are immobilized on a support and those with which the necessary amounts of the secondary antibody and the substrate are enclosed.
The aforementioned reagents can be used as agents for diagnosis of Chlamydia pneumoniae infections. Probes and Primers for Detection and/or Measurement of Chlamydia pneumoniae Gene
DNA encoding the Chlamydia pneumoniae 53 kDa antigenic polypeptide has the base sequence of SEQ ID NO: 3.
The probes and primers of the invention comprise DNA containing any one of
(a) a DNA containing a sequence of at least 10 consecutive bases in the DNA of SEQ ID NO: 3,
(b) a DNA complementary to DNA (a), or
(c) a DNA having at least 90% homology to DNA (a) or (b).
The length of the base sequence of the probes and primers is preferably 10-50 bp, more preferably 15-20 bp.
Specific examples of the probes and primers of the invention include a DNA comprising the base sequence of SEQ ID NO: 19 and a DNA comprising the base, sequence of SEQ ID NO: 20.
The probes and primers of the invention can be synthesized easily with a commercially available DNA synthesizer. DNA synthesizers are commercially available from Applied Biosystems and the like. Alternatively, the probes and primers of the invention can be prepared by chemically synthesizing a short DNA fragment and synthesizing a long DNA fragment by PCR using the short DNA as a primer.
The probes and primers of the invention include those prepared by labeling such DNAS.
Exemplary labels include chemical compounds such as biotin, avidin, streptoavidin, digoxigenin and the like; enzymes such as alkaline phosphatase, luciferase, peroxidase, xcex2-galactosidase and the like; and fluorescent compounds such as fluorescein and the like. Biotin may be bound to the probes by, for example, adding biotinated deoxyuridine 5xe2x80x2-triphosphate to the probes in the presence of a terminal transferase. A kit containing a terminal transferase and biotinated deoxyuridine 5xe2x80x2-triphosphate can be purchased from Boehringer Mannheim. In the case where a label other than biotin is to be bound, a commercially available kit can also be used. Such a kit can be purchased from Takara Shuzo Co., Ltd and TOYOBO CO., LTD. Alternatively, the label may be bound by a method described in xe2x80x9cMolecular Cloningxe2x80x9d.
If desired, radioactive isotopes can be used as labels. In this case, (xcex3-32p)DATP is added to the probes and primers in the presence of T4 polynucleotide kinase. A general procedure of labeling with a radioactive isotope is described in xe2x80x9cMolecular Cloningxe2x80x9d. T4 polynucleotide kinase can be purchased from TOYOBO CO., LTD. and (xcex3-32P)DATP from Amersham.
RNAs corresponding to the base sequences of the probes and primers of the invention, that is, nucleic acids in which thymine is replaced with uracil in the base moiety and in which deoxyriboses are replaced with riboses in the sugar chain, can be used as the probes and primers of the invention instead of the aforementioned probes and primer comprising DNAS as structural units. These probes and primers comprising RNAs as structural units can be used in the method and reagents for detection and/or measurement of the invention.
Chlamydia pneumoniae gene is detected and/or measured by, for example, separating DNA in a sample on the basis of the difference in molecular weight by elecrophoresis, transferring the obtained DNA to a nitrocellulose filter, nylon membrane filter or the like for its identification, adding the labeled probe of the invention, and detecting and/or measuring the label. This method is called the Southern blotting technique and its general procedure is described in xe2x80x9cMolecular Cloningxe2x80x9d.
Chlamydia pneumoniae gene is detected and/or measured with the primer of the invention by, for example, the PCR method which was described above. The method for detecting and/or measuring Chlamydia pneumoniae gene by PCR using the primer of the invention comprises the following steps.
(i) A buffer containing the primer of the invention, DNA polymerase, DATP, dCTP, dGTP and dTTP is added to a sample containing DNA and the mixture is heated.
(ii) The reaction solution is cooled, held at a constant temperature and heated.
(iii) Step (ii) is repeated.
(iv) The DNA contained in the reaction solution is detected and/or measured.
The DNA-containing sample to be used in step (i) may be nucleic acids as extracted from tunica mucosa pharyngsis of a patient.
The DNA polymerase to be used in step (i) may be a Taq polymerase, which can be purchased from TOYOBO CO., LTD.
In step (i), the mixture is heated by, for example, leaving it to stand at 90-100xc2x0 C. for 0.5-10 minutes.
In step (ii), the reaction solution is cooled by, for example, leaving it to stand at 45-65xc2x0 C. for 0.5-5 minutes, held at a constant temperature by, for example, at 70-80xc2x0 C. for 1-10 minutes, heated by, for example, leaving it to stand at 90-100xc2x0 C. for 0.5-5 minutes.
The heating in step (i), and cooling, holding at a constant temperature and heating in step (ii) can be carried out by using a DNA thermal cycler(copyright) (Perkin-Elmer Cetus).
Step (iii) may be repeated any number of times, preferably about 30 times.
The DNA contained in the reaction solution is detected and/or measured in step (iv) by, for example, electrophoresing the reaction solution with an agarose gel containing ethidium bromide, and thereby separating the DNA in the reaction solution on the basis of the difference in molecular weight and irradiating the agarose gel with UV light. If the primer of the invention is a labeled one, DNA is detected and/or measured with the aid of the label.
In another embodiment of the invention, after steps (i)-(iii), the primer of the invention may be replaced with one having another base sequence and steps (i)-(iii) are repeated, followed by step (iv).
An exemplary reagent for detection and/or measurement of Chlamydia pneumoniae gene according to the invention is an aqueous solution of the probe or primer of the invention which is packed frozen in a plastic container.